Initiation and Regulation of Fibrinolysis in Human Plasma at the Plasminogen Activator Level

Abstract

The initiation and regulation of fibrinolysis has been studied by reconstitution of fibrinolytic activity in human plasma in vitro. Depletion of tissue plasminogen activator (tPA) antigen by immunoadsorption of human plasma with anti-tPA Ig Sepharose 4B leads to total loss of spontaneous fibrinolytic activity determined by lysis of a thrombin-induced clot. Addition of physiological concentrations of purified tPA to tPA-depleted plasma restores fibrinolytic activity as a function of the length of time between tPA addition and clotting. Addition of free tPA to tPA-depleted plasma followed by immediate clotting results in a high rate of fibrinolysis. In contrast, when free tPA is allowed to incubate in plasma for 10 to 60 minutes prior to clot formation, the fibrinolytic activity of tPA is gradually lost. The loss of tPA-induced fibrinolytic activity in unclotted plasma is accompanied by decreased partitioning of tPA antigen into fibrin after clotting and is kinetically correlated with the formation of a 100 kilodalton (kDa) tPA complex as demonstrated by SDS-gel electrophoresis and fibrin-agar zymography. These results suggest that free tPA is susceptible to complexation by the plasma inhibitor in the absence of a clot. Fibrin formation renders tPA relatively inaccessible to inhibition. The tPA antigen isolated from stored plasma consists mainly of 100 kDa activity in SDS-gel electrophoresis and zymography, indicating that the tPA complex is resistant to dissociation by SDS. Upon rezymography of the sliced gel, only a 60 kDa tPA activity is found, suggesting that the activity at 100 kDa is at least partly due to free tPA dissociated from the complex during the first zymography. Conversion of tPA complex to enzymatically active free tPA also occurs with brief SDS exposure followed by incubation in the presence of excess Triton X-100 or by hydroxylamine treatment. These results reconcile the apparent discrepancy of the 100 kDA inhibitor-tPA complex manifesting plasminogen activation activity during zymography. The plasma tPA-inhibitor complex is precipitated strongly by antisera against plasminogen activator inhibitors (PAIs) of human Hep G2 hepatoma and HT-1080 fibrosarcoma cells and weakly by antiserum against bovine aortic endothelial cell PAI but not by antiserum against a placental PAI (PAI-2) suggesting that the plasma inhibitor is immunologically related to Hep G2, HT-1080 and possibly endothedial cell PAIs. Based on the above findings, a simple model for the initiation and regulation of plasma fibrinolysis at the PA level has been formulated.